Several subtypes of the α2δ subunit of calcium channel have been cloned (Angeloni et al. Mol. Cell. Probes 14:53–54, 2000; Gao et al., J. Biol. Chem. 275:12237–12242, 2000; and PCT Application WO 99/23519).
Neuroblastoma cell membranes contain N-type calcium channels and neuroblastoma cells have been used as a model for neuronal differentiation (Bruhn, et al. Endocrinology 137:572–9, 1996; Gotti, et al. Differentiation (Berlin) 34:144–55, 1987; Hogg et al. Pharmacol., 312:257–261, 1996; and Kurata, et al. FEBS Lett., 321:201–4, 1993). 5-Bromo-2′-deoxyuridine (BrdU) induces morphological and functional differentiation of neuroblastoma cells, resulting in an increase of neurotransmitter receptors and the release of neurotransmitters (Clementi, et al. Adv. Exp. Med. Biol. 296:91–102, 1991). [125I]ω-conotoxin binding sites were increased in the differentiated neuroblastoma IMR32 cells, indicating that the N-type calcium channels were increased in the cells (Carbone et al., Pfluegers Arch., 416:170–9, 1990). Recently, Western blot analysis has shown that the β1b subunit of calcium channels is the predominant isoform expressed in IMR32 cells (McEnery, et al. FEBS Lett. 420: 74–78, 1997).
Gabapentin (GBP) is an anticonvulsant that has shown usefulness in the treatment of neuropathic pain (Backonjy, M. et al. J. Am. Med. Assoc. 280:1831–1836, 1998; Laird and Gidal, Ann. Pharmacother. 34:802–807, 2000; and Rowbotham et al. M., J. Am. Med. Assoc., 280:1837–1842, 1998). GBP inhibits neurotransmitter release (Dooley, et al. Neurosci. Lett. 280:107–110, 2000) and inhibits calcium currents in brain neurons (Fink, et al. Br. J. Pharmacol. 130:900–906, 2000; Larid and Gidal, supra; and Stefani et al. Neuropharmacology 37:83–91, 1998). Interestingly, a high-affinity binding site for GBP was found in brain tissue and the target protein was identified as the α2δ subunit of subunit of calcium channels (Brown and Gee, J. Biol. Chem., 273:25458–25465, 1998; Dissanayake, et al. Br. J. Pharmacol., 120:833–840, 1997; and Gee et al., J. Biol. Chem. 271:5768–76, 1996). An autoradiographic binding study showed that the GBP binding site was widely distributed in rat brain areas such as frontal cortex, striatum, hippocampus and cerebellum (Hill and Woodruff, Eur. J. Pharmacol. Mol. Pharmacol. Sect., 244:303–9, 1993; Thurlow et al. Br. J. Pharmacol., 118;457–465, 1996). Therefore compounds that compete with Gabapentin binding to the α2δ subunit should be useful as anticonvulsants and in the treatment of neuropathic and chronic pain.